HIV-1 maturation occurs via temporally regulated proteolysis of the Gag and Gag-Pol polyproteins at specific sites, leading to formation of a functional HIV-1 core. We have recently shown that 3-0-{3',3'- dimethylsuccinyl}-betulinic acid (DSB) blocks HIV-1 replication through a novel mechanism of action. DSB specifically inhibits the final step in HIV-1 Gag processing-cleavage at the CA-p2 junction. Inhibition requires the drug to be present at the time of HIV-1 assembly, indicating its action at a late stage of replication. However, the compound does not exhibit activity against the HIV-1 protease (PR) in vitro using peptides or recombinant Gag protein as substrates. HIV-1 acquires partial resistance to DSB by missense mutations at the CA-p2 junction, and mutations in PR that render HIV-1 resistant to PR inhibitors confer little to no resistance to DSB. We hypothesize that DSB associates with the CA-p2 junction during HIV-1 assembly or maturation, resulting in delayed PR cleavage of this site. Here we propose to further define the mechanism and consequences of DSB action through two Specific Aims: (1) to identify the molecular target of DSB; and (2) to determine the spectrum of resistance of HIV-1 to DSB and the fitness cost associated with resistance. These studies will precisely define the HIV-1 target of DSB as a potent small-molecule inhibitor of HIV-1 replication, thus providing information that will guide the design of the next generation of HIV-1 maturation inhibitors. Because DSB is a promising antiviral therapy that is currently being developed by Panacos Pharmaceuticals, a comprehensive analysis of HIV-1 resistance to DSB will serve to inform the human efficacy trials of DSB that will likely be initiated in the near future.